Rapidly evolving genotyping techniques are being used in an attempt to identify the genetic basis of hereditary diseases and to establish a genotype/phenotype correlation, which in turn may allow for a more predictive molecular diagnosis. Yet, most hereditary diseases are genetically very complex and often involve multiple allelic combinations, which may not always show a disease-specific phenotype. In such cases, genotyping of all allelic variants is necessary to be able to distinguish between e.g. non-carriers and disease carriers. Only knowledge of the exact genotype of an individual will allow for an accurate prognosis of the inheritance pattern, an accurate prediction of disease-related symptoms to be expected as the disease progresses as well as an accurate therapy for the individual.
Such diseases include all genetic diseases based on the multiplication of a part of the genome due to unequal crossover events between homologous chromosomes, leading to homologous sequence clusters (duplications) and deletions on a chromosome (FIG. 1). Such diseases include for example spinal muscular atrophy (SMA), diverse microtriplications, and thalassemia. Thalassemia, for example, is a common genetic disorder, which leads in its most moderate form to a hypochromic microcytic anaemia due to impaired hemoglobin formation. The clinical outcome of more severe cases leads to very severe anemia or hydrops fetalis. Depending on the underlying genetic defects, thalassemia is classified into β-thalassemia and α-thalassemia. In β-thalssemia the majority of cases are due to point or raster mutations in the β-globin-locus on chromosome 11, which contains a single gene encoding the β-globin chains. The α-thalassemias, which are classified into α-thalassemia, α+-thalassemia and α0-thalassemia, are mainly the result of deletions on chromosome 16, which contains at its telomeric region two highly homologous and closely linked genes (α1- and α2-gene) encoding the α globin chains. The duplicated alpha-globin genes α1 and α2 are embedded within two markedly homologous regions that extend for approximately 4 kb. During meiosis, misalignment of chromosome homologs followed by reciprocal recombination at three highly homologous segments, named X, Y, and Z, results in various deletion-duplication events (FIG. 2).
The causes of α-thalassemia, α+-thalassemia and α0-thalassemia, are the deletion or dysfunction of one or both α1 and α2 genes, respectively.
Deletional α+-thalassemia results from loss of one of the two α-globin genes (αα/−α), e.g. by reciprocal recombination between the Z region, which are 3.7 kb apart, or between the X region, 4.2 kb apart, giving rise to the −α3.7kb and −α4.2kb deletion, respectively. FIG. 2 is a schematic illustration of the unequal crossover events leading to those deletions (filled squares represent the functional active genes α1 and α2 of the alpha globin gene cluster and blank squares represent the homologues X-, Y- and Z-boxes and one of the two pseudoglobingenes, i.e. Ψα1). Heterozygote carriers of α+-thalassemia resulting from the combination of a deletional α+-thalassemia (−α) and a wt (αα) (also known as α-thalassemia silent carrier (−α/αα)) may have a silent hematologic phenotype instead homozygote carriers ((−α/−α)) present a moderate thalassemia-like hematologic picture (see e.g. Herklotz et al, Ther Umsch, 2006, 63 (1), p. 35).
α°-Thalassemia may be caused by extended deletions varying from 5.2 kb to 25 kb and more resulting in deletion or dysfunction of both, the α1 and the α2 genes (homozygotes (−−/−−) or heterozygotes (αα/−−)), e.g. −αSEA, −αTAI, −αFIL, −αMED, −(α)20.5kb. About 30 different such α°-thalassemia deletions have been reported to date.
The outcomes of the α-thalassemias are manifold and the severity is correlated with the number of affected α-globin loci, i.e. the exact nature of the gene deletion, as illustrated in FIG. 3 (filled boxes: α-globin gene(s) present, blank boxes: α-globin gene(s) deleted).
The phenotypes of α-thalassemia have two clinically significant forms, which are Hb Bart hydrops fetalis (Hb Bart) syndrome and hemoglobin H (HbH) disease. In Hb Bart, all four α-globin alleles are deleted or inactivated (−−/−−). It is the most severe form and is characterized by fetal onset of generalized edema, with death in the neonatal period being almost inevitable. HbH disease is a result of deletion or dysfunction of three of the four α-globin alleles (−−/−α). It is characterized by microcytic hypochromic hemolytic anemia, hepatosplenomegaly, mild jaundice, and sometimes bone and heart changes.
The milder forms of thalassemia ((−α/−α), (−−/αα) lead to hematologic changes, usually without any clinical symptoms. However, for the so called “silent” thalassemia the exact diagnosis is still very important (αα/−α).
It is estimated that there are at least 200 million people affected worldwide. In addition, 300′000-400′000 severely affected infants are born every year, more than 95% of which occur in Asia, India, and the Middle East.
Current testing for α-thalassemia is based on an algorithm of exclusion-diagnosis (i.e. to exclude iron deficiency, β-thalassemia, the haemoglobinopathies and hemolytic anemia), which requires a wide range of procedures such as hematologic testing of red blood cell indices, peripheral blood smear, supravital staining to detect RBC inclusion bodies, qualitative and quantitative hemoglobin analysis, bone marrow examination, and in vitro synthesis of radioactive-labeled globin chains in affected individuals. Final proof for the presence of an α-thalassemia is obtained using biomolecular diagnostics (Huber et al., Swiss Medical Forum, 2004). This includes polymerase chain reaction (gap-PCR) amplification of the normal α1 and α2 or hybrid α2/α1 globin genes (Chang et al., Blood, 1991, 78 (3), 853; Ko et al., Hum Genet, 1992, 88 (3), 245), enzyme-linked immuno-sorbent assays (ELISA) for the detection of zeta-globin chains in circulation (Ausavarungnirun et al., Am J Hematol, 1998, 57 (4), 283) and hybridization assays with α-strips.
The Real-time Quantitative PCR technique (RT-PCR) has been applied for different investigations including pathogen detection, allelic discrimination, gene expression and gene regulation (Bowie et al., Clin Chem, 1994, 40 (12), 2260; Das et al., Br J Cancer, 2000, 82 (10), 1682; Fujii et at., Mutat, 2000, 15 (2), 189), as well as the detection of duplications and deletions, e.g. in Duchenne and Becker muscular dystrophies, cystic fibrosis and neuroblastomas (Joncourt et al., Hum Mutat 2004, 23 (4): 385), detection of exon deletions within an entire gene (CFTR) by relative quantification on the LightCycler (Schneider M. et al., American Association for Clinical Chemistry 2006, 52: 11) Quantification of MYCN, DDX1 and NAG gene copy number in neuroblasotma using a real-time quantitative PCR assay (De Preter K. et al., Mod Pathol 2002, 15 (2): 159-166). However, while RT-PCR has also been applied for the detection of α-thalassemia (Armour et al., Hum Mutat, 2002, 20 (5), 325), current methods only allow for detection of several restricted mutations such as the southeast Asian type deletion, or a group of e.g. three different deletions (−α3.7kb, −αSEA and −αMED).
Clearly, current technologies are labor-intensive and/or time-consuming and in case of hereditary diseases linked to repeat gene clusters, i.e. multiple genes, such as α-thalassemia, may still not provide an accurate analysis of all variants of the diseases.
Thus, there is a great need for a general, rapid and efficient screening method, which is completely standardized and suitable for routine laboratory, which allows the differential quantitative detection of multiple homologous gene loci underlying a specific hereditary disease, such as the ones mentioned hereinabove.
Applicants have now designed a new screening method using multiple primer sets performing only one single RT-PCR run, which enables classification of the genotype of an individual affected by such a hereditary disease. Quantification of each of the amplicated gene regions in reference to a control, preferably one or several endogenous control (reference gene), and particularly the relationship between each of the amplicated regions allows the well-defined identification of the genotype of the individual. In case of an aberrant genotype, subsequent analysis, e.g. sequencing, allows to further characterize the exact nature and location of the mutation.
The new method according to the invention is applicable to any genetic disorder based on multiple homologous gene loci. For example, applicants have shown that the new screening method allows for a clear classification of the genotype of the α-thalassemia of any patient performing one single RT-PCR run. The quantification of each of the amplificated α-globin-gene regions and particularly the relationship between those regions will allow for the determination of the real prevalence of α-thalassemia detecting all carriers, which may otherwise be subject to mis- or nondiagnosis. This is a particularly important tool with respect to genetic counseling in general and/or prenatal diagnosis.